This grant has supported our major effort to characterize farnesylated proteins. Our study during the current funding period uncovered novel features of Rheb G-protein. We have shown that Rheb represents a unique family of the Ras-superfamily G-proteins that is highly conserved in yeast, Drosophila and human. Our studies in Schizosaccharomyces pombe established that Rheb is essential and plays a role in the regulation of cell cycle and cell growth. Recent studies on Drosophila Rheb suggested that Rheb may play a role in the insulin/RS6K signaling. Consistent with this idea, we have shown that Rheb is capable of activating S6K in mammalian cells. In this application, we propose first to evaluate the model that Rheb is a component of TOR signaling. We will utilize S. pombe, Drosophila and mammalian cells to examine interaction of Rheb with TOR/S6Kfunctions. In addition, interaction of Rheb with TSC will be evaluated. TSC2 protein is of particular interest, as this tumor suppressor gene product contains a GAP (GTPase activating protein) domain raising the possibility that TSC2 functions as a GAP for Rheb. In the second specific aim, we propose to carry outscreens and selections to identify genes affecting Rheb function. The screens will be carried out in yeast, since yeast provides a simple and powerful system to evaluate significance of any genes identified from the screen. Dominant negative Rheb mutants may lead us to the identification of GEF (GDP/GTP exchange factor) for Rheb. Yeast two-hybrid assays using the wild type and Rheb effector domain mutants will be carried out to identify downstream effectors of Rheb. We will also make use of a heterologous S. cerevisiaesystem to gain insight into genes affecting S. pombe Rheb. S. pombe genes obtained will be used to identify and characterize Drosophila and mammalian counterparts. Finally, we will attempt to inhibit S6K activation mediated by Rheb activation. We will reexamine a previous observation that farnesyl transferase inhibitor(FTI) is capable of blocking activation of S6K in mammalian cells. This study will be followed by the characterization of TSC mutant cells for possible activation of Rheb as well as their sensitivity to FTI. These experiments should have significant implication for understanding how Rheb affects Torsignalng. The study will also be of importance for cancer therapy, as our study may reveal the ability of FTIto inhibit over-activation of RhebFFOR/S6K in tumor cells.